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Journal of Integrative Agriculture  2022, Vol. 21 Issue (2): 504-520    DOI: 10.1016/S2095-3119(21)63644-4
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection
XIE Xing1, HAO Fei1, WANG Hai-yan1, PANG Mao-da2, GAN Yuan1, LIU Bei-bei1, ZHANG Lei1, WEI Yan-na1, CHEN Rong1, ZHANG Zhen-zhen1, BAO Wen-bin3, BAI Yun1, SHAO Guo-qing1, XIONG Qi-yan1, FENG Zhi-xin1
1 Key Laboratory for Veterinary Bio-Product Engineering of Ministry of Agriculture and Rural Affairs, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China
2 Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China
3 College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China
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Abstract  Primary porcine tracheal epithelial cells (PTECs) are an appropriate model for studying the molecular mechanism of various porcine respiratory diseases, including swine-origin mycoplasmas, which are isolated from respiratory tract of pigs and mainly found on the mucosal surface surrounding swine trachea.  However, the short proliferation ability of primary PTECs greatly limits their lifespan.  In this study, primary PTECs were carefully isolated and cultured, and immortal PTECs were constructed by transfecting primary PTECs with the recombinant constructed plasmid pEGFP-hTERT containing human telomerase reverse transcriptase (hTERT).  Immortal PTECs (hTERT-PTECs) maintained both the morphological and functional characteristics of primary PTECs, as indicated by the expression of cytokeratin 18, cell-cycle analysis, proliferation assay, Western blotting, telomerase activity assay, karyotype analysis and quantitative RT-PCR.  Compared to primary PTECs, hTERT-PTECs had an extended replicative lifespan, higher telomerase activity, and enhanced proliferative activity.  In addition, this cell line resulted in a lack of transformed and grown tumors in nude mice, suggesting that it could be safely applied in further studies.  Moreover, hTERT-PTECs were vulnerable to all swine-origin mycoplasmas through quantitative analysis as indicated by 50% color changing unit (CCU50) calculation, and no significant differences of adhesion ability between primary and immortal PTECs were observed.  For the representative swine mycoplasma Mycoplasma hyopneumoniae (Mhp), except for DNA copies quantitative real-time PCR assay, indirect immunofluorescence assay and Western blotting analysis also depicted that hTERT-PTECs was able to adhere to different Mhp strains of different virulence.  In summary, like primary PTECs, hTERT-PTECs could be widely used as an adhesion cell model for swine-origin mycoplasmas and in infection studies of various porcine respiratory pathogens.  
Keywords:  porcine tracheal epithelial cells (PTECs)       hTERT-PTECs       swine-origin mycoplasmas       adhesion       cell model 

Received: 02 September 2020   Accepted: 01 February 2021
Fund: This work was supported by the National Natural Science Foundation of China (31800161, 31700157, 31800160, 31900159, and 31770193), the Natural Science Foundation of Jiangsu Province, China (BK20180297 and BK20170600), and the Independent Research Project Program of Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base, Ministry of Science and Technology, China (2019sy004).
About author:  XIE Xing, Tel: +86-25-84390881, E-mail:; Correspondence FENG Zhi-xin, Tel: +86-25-84391320, E-mail:; XIONG Qi-yan, E-mail:

Cite this article: 

XIE Xing,  HAO Fei, WANG Hai-yan, PANG Mao-da, GAN Yuan, LIU Bei-bei, ZHANG Lei, WEI Yan-na, CHEN Rong, ZHANG Zhen-zhen, BAO Wen-bin, BAI Yun, SHAO Guo-qing, XIONG Qi-yan, FENG Zhi-xin. 2022. Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection. Journal of Integrative Agriculture, 21(2): 504-520.

Abraham G, Zizzadoro C, Kacza J, Ellenberger C, Abs V, Franke J, Schoon H A, Seeger J, Tesfaigzi Y, Ungemach F R. 2011. Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions. BMC Veterinary Research, 7, 26.
Blanchard B, Vena M M, Cavalier A, Le Lannic J, Gouranton J, Kobisch M. 1992. Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae. Veterinary Microbiology, 30, 329–341.
Bodnar A G, Ouellette M, Frolkis M, Holt S E, Chiu C P, Morin G B, Harley C B, Shay J W, Lichtsteiner S, Wright W E. 1998. Extension of life-span by introduction of telomerase into normal human cells. Science, 279, 349–352.
Calaf G M, Roy D. 2008. Cancer genes induced by malathion and parathion in the presence of estrogen in breast cells. International Journal of Molecular Medicine, 21, 261–268.
Chen X, Zhang Q, Bai J, Zhao Y, Wang X, Wang H, Jiang P. 2017. The nucleocapsid protein and non-structural protein 10 of highly pathogenic porcine reproductive and respiratory syndrome virus enhance CD83 production via NF-kappaB and Sp1 signaling pathways. Journal of Virology, 91, e00986–e01003.
Ferrarini M G, Siqueira F M, Mucha S G, Palama T L, Jobard E, Elena-Herrmann B, At R V, Tardy F, Schrank I S, Zaha A, Sagot M F. 2016. Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling. BMC Genomics, 17, 353.
Furr P M, Taylor-Robinson D. 1993. Factors influencing the ability of different mycoplasmas to colonize the genital tract of hormone-treated female mice. International Journal of Experimental Pathology, 74, 97–101.
Ha Thi B M, Campolmi N, He Z, Pipparelli A, Manissolle C, Thuret J Y, Piselli S, Forest F, Peoc’h M, Garraud O, Gain P, Thuret G. 2014. Microarray analysis of cell cycle gene expression in adult human corneal endothelial cells. PLoS ONE, 9, e94349.
Hahn W C. 2002. Immortalization and transformation of human cells. Molecules and Cells, 13, 351–361.
He Y L, Wu Y H, He X N, Liu F J, He X Y, Zhang Y. 2009. An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer. Theriogenology, 71, 1417–1424.
Hsu T, Minion F C. 1998. Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin. Infection and Immunity, 66, 4762–4766.
Kobisch M, Friis N F. 1996. Swine mycoplasmoses. Revue Scientifique et Technique-Office International des Epizooties, 15, 1569–1605.
Lam H C, Choi A M, Ryter S W. 2011. Isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface. Journal of Visualized Experiments, 48, 1–4.
Lamontagne J, Mell J C, Bouchard M J. 2016. Transcriptome-Wide analysis of Hepatitis B virus-mediated changes to normal hepatocyte gene expression. PLoS Pathogen, 12, e1005438.
Lange A W, Keiser A R, Wells J M, Zorn A M, Whitsett J A. 2009. Sox17 promotes cell cycle progression and inhibits TGF-beta/Smad3 signaling to initiate progenitor cell behavior in the respiratory epithelium. PLoS ONE, 4, e5711.
Leal Zimmer F M A, Moura H, Barr J R, Ferreira H B. 2019. Intracellular changes of a swine tracheal cell line infected with a Mycoplasma hyopneumoniae pathogenic strain. Microbial Pathogenesis, 137, 103717. 
Leigh S A, Evans J D, Branton S L, Collier S D. 2008. The effects of increasing sodium chloride concentration on Mycoplasma gallisepticum vaccine survival in solution. Avian Diseases, 52, 136–138.
MacHugh D E, Taraktsoglou M, Killick K E, Nalpas N C, Browne J A, Park S D E, Hokamp K, Gormley E, Magee D A. 2012. Pan-genomic analysis of bovine monocyte-derived macrophage gene expression in response to in vitro infection with Mycobacterium avium subspecies paratuberculosis. Veterinary Research, 43, 25.
Maes D, Sibila M, Kuhnert P, Segales J, Haesebrouck F, Pieters M. 2018. Update on Mycoplasma hyopneumoniae infections in pigs: Knowledge gaps for improved disease control. Transboundary Emerging Diseases, 65(Suppl. 1), 110–124.
Mathieu-Denoncourt A, Letendre C, Auger J P, Segura M, Aragon V, Lacouture S, Gottschalk M. 2018. Limited Interactions between Streptococcus suis and Haemophilus parasuis in in vitro co-infection studies. Pathogens, 7, 7.
Mayer A K, Bartz H, Fey F, Schmidt L M, Dalpke A H. 2008. Airway epithelial cells modify immune responses by inducing an anti-inflammatory microenvironment. European Journal of Immunology, 38, 1689–1699.
Miyazawa K, Hondo T, Kanaya T, Tanaka S, Takakura I, Itani W, Rose M T, Kitazawa H, Yamaguchi T, Aso H. 2010. Characterization of newly established bovine intestinal epithelial cell line. Histochemistry and Cell Biology, 133, 125–134.
Nalpas N C, Park S D, Magee D A, Taraktsoglou M, Browne J A, Conlon K M, Rue-Albrecht K, Killick K E, Hokamp K, Lohan A J, Loftus B J, Gormley E, Gordon S V, MacHugh D E. 2013. Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro. BMC Genomics, 14, 230.
Nicholson T L, Brockmeier S L, Sukumar N, Paharik A E, Lister J L, Horswill A R, Kehrli M E, Loving Jr C L, Shore S M, Deora R. 2017. The Bordetella Bps polysaccharide is required for biofilm formation and enhances survival in the lower respiratory tract of swine. Infection and Immunity, 85, e00261–e00278.
Pieters M, Daniels J, Rovira A. 2017. Comparison of sample types and diagnostic methods for in vivo detection of Mycoplasma hyopneumoniae during early stages of infection. Veterinary Microbiology, 203, 103–109.
Ramirez R D, Sheridan S, Girard L, Sato M, Kim Y, Pollack J, Peyton M, Zou Y, Kurie J M, Dimaio J M, Milchgrub S, Smith A L, Souza R F, Gilbey L, Zhang X, Gandia K, Vaughan M B, Wright W E, Gazdar A F, Shay J W, Minna J D. 2004. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Research, 64, 9027–9034.
Randell S H, Walstad L, Schwab U E, Grubb B R, Yankaskas J R. 2001. Isolation and culture of airway epithelial cells from chronically infected human lungs. In Vitro Cellular & Developmental Biology (Animal), 37, 480–489.
Raymond B B A, Turnbull L, Jenkins C, Madhkoor R, Schleicher I, Uphoff C C, Whitchurch C B, Rohde M, Djordjevic S P. 2018. Mycoplasma hyopneumoniae resides intracellularly within porcine epithelial cells. Scientific Reports, 8, 17697.
Sreenivasan C C, Thomas M, Antony L, Wormstadt T, Hildreth M B, Wang D, Hause B, Francis D H, Li F, Kaushik R S. 2019. Development and characterization of swine primary respiratory epithelial cells and their susceptibility to infection by four influenza virus types. Virology, 528, 152–163.
Su F, Liu X, Liu G, Yu Y. Wang Y, Jin Y, Hu G, Hua S, Zhang Y. 2013. Establishment and evaluation of a stable cattle type II alveolar epithelial cell line. PLoS ONE, 8, e76036.
Tompkins D H, Besnard V, Lange A W, Keiser A R, Wert S E, Bruno M D, Whitsett J A. 2011. Sox2 activates cell proliferation and differentiation in the respiratory epithelium. American Journal of Respiratory Cell and Molecular Biology, 45, 101–110.
Wang H, He L, Liu B, Feng Y, Zhou H, Zhang Z, Wu Y, Wang J, Gan Y, Yuan T, Wu M, Xie X, Feng Z. 2018. Establishment and comparison of air-liquid interface culture systems for primary and immortalized swine tracheal epithelial cells. BMC Cell Biology, 19, 10.
Wang J, Hu G, Lin Z, He L, Xu L, Zhang Y. 2014. Characteristic and functional analysis of a newly established porcine small intestinal epithelial cell line. PLoS ONE, 9, e110916.
Xie X, Gan Y, Pang M, Shao G, Zhang L, Liu B, Xu Q, Wang H, Feng Y, Yu Y, Chen R, Wu M, Zhang Z, Hua L, Xiong Q, Liu M, Feng Z. 2018. Establishment and characterization of a telomerase-immortalized porcine bronchial epithelial cell line. Journal of Cellular Physiology, 233, 9763–9776.
Xie X, Pang M, Liang S, Yu L, Zhao Y, Ma K, Kalhoro D H, Lu C, Liu Y. 2015. Establishment and characterization of a telomerase-immortalized canine bronchiolar epithelial cell line. Applied Microbiology and Biotechnology, 99, 9135–9146.
Xiong Q, Wang J, Ji Y, Ni B, Zhang B, Ma Q, Wei Y, Xiao S, Feng Z, Liu M, Shao G. 2016. The functions of the variable lipoprotein family of Mycoplasma hyorhinis in adherence to host cells. Veterinary Microbiology, 186, 82–89.
Xiong Q, Wei Y, Feng Z, Gan Y, Liu Z, Liu M, Bai F, Shao G. 2014. Protective efficacy of a live attenuated Mycoplasma hyopneumoniae vaccine with an ISCOM-matrix adjuvant in pigs. Veterinary Journal, 199, 268–274.
Yang W, Lambertz R L O, Punyadarsaniya D, Leist S R, Stech J, Schughart K, Herrler G, Wu N H, Meng F. 2017. Increased virulence of a PB2/HA mutant of an avian H9N2 influenza strain after three passages in porcine differentiated airway epithelial cells. Veterinary Microbiology, 211, 129–134.
Yoon J H, Gray T, Guzman K, Koo J S, Nettesheim P. 1997. Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix. American Journal of Respiratory Cell and Molecular Biology, 16, 724–731.
Yu Y, Liu M, Hua L, Qiu M, Zhang W, Wei Y, Gan Y, Feng Z, Shao G, Xiong Q. 2018. Fructose-1,6-bisphosphate aldolase encoded by a core gene of Mycoplasma hyopneumoniae contributes to host cell adhesion. Veterinary Research, 49, 114.
Zhang L, Huang Y, Wang Z, Luo X, Zhang H, Du Q, Chang L, Zhao X, Tong D. 2017. Establishment and characterization of a telomerase immortalized porcine luteal cells. Theriogenology, 94, 105–113.
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